PRE-IMPLANTATION GENETIC DIAGNOSIS

ANDASSISTED REPRODUCTIVE

TECHNOLOGYIN HAEMOPHILIA

DR PENELOPE FOSTER

WHAT IS PGD ?

• early embryo diagnosis• allows identification of gender or abnormal

gene• can select which embryos to transfer to

patient• an alternative to antenatal testing and

termination of affected pregnancy

TECHNIQUE OF PGD

.standard IVF cycle

.biopsy of 1 or 2 cells from day 3 embryo

.diagnostic testing on biopsied cells

.selection of embryos for transfer

PGD IN HAEMOPHILIAOPTIONS (1)

GENDER SELECTIONIf male partner has haemophilia:(all male offspring unaffected, all female offspring carriers)Gender selection enables transfer of male embryos only,

excludes all carriersIf female partner is carrier:(50% male offspring affected, 50% female offspring

carriers)Gender selection enables transfer of female embryos only,but 50% discarded male embryos unaffected, 50% of

transferred female embryos carriers

PGD IN HAEMOPHILIA

OPTIONS (2)SPECIFIC GENE DETECTION(identifies embryos with the X chromosome

mutation assoc with haemophilia) .more embryos available for transfer (~60%).avoids discarding unaffected male embryos.avoids transfer of carrier female embryos

GENDER SELECTION - FISHFLUORESCENT IN-SITU HYBRIDISATION

.detects the presence and number of particular chromosomes inc Xand Y

.single cell from embryo fixed to slide

.apply FISH probes.labelled DNA probes which bind to complementary sequences on

specific chromosomes.probes labelled with coloured fluorochromes.coloured spots indicate presence of sequence

.8-probe FISH – chromosomes 4,13,16,18,21,22,X,Y

.select euploid XX or XY embryos for transfer

.only ~15% of embryos suitable for embryo transfer

.60% patients will not have embryo transfer

13, 16, 18, 21, 22 X, Y, 4

FISH ON BLASTOMERESFISH ON BLASTOMERES

XX XY

PGD FOR SPECIFIC GENE DETECTION

.looking for the presence or absence of the haemophilia mutation in each embryo tested

.2 cells from each embryo. DNA amplification (by PCR).fragment analysis on DNA sequencer.analyse polymorphic markers along the F8 gene.inclusion of informative markers (belt and braces).individualised tests for each couple.significant time and effort required for each test

Markers AllelesDXS1073 124, 126, 128DXS8061 139, 145, 147Factor VIII wt, mut

AMEL 111 = X c.some

117 = Y c.some

Unaffected female Unaffected Male

128147wt

126139mut

124145wt

124145wt

126139mut

Carrier Female

Affected male

MotherHusband

128147wt

MOTHER

AFFECTED MALE

RESULTS OF PCR ANALYSIS FOR HAEMOPHILIA A

PGD options in HaemophiliaIf man has haemophilia:

gender selection (for male embryos)but with 8-probe FISH

only ~ 10 -15% embryos suitable for transferin 60% cycles there will be no transfergene detectionno value as all males unaffected, all females carriers

If female is carrier:gender selection (for female embryos)but with 8-probe FISH only ~10 -15% embryos suitable for transferin 60% cycles there will be no transfer½ discarded males unaffected, ½ transferred females carriersgene detectionselect unaffected male embryo or non-carrier female embryos88% of cycles will have embryo transfer

PGD OUTCOMES (MIVF data 1997 - 2008)

21.030.8IMP. RATE

24.833.3% CLIN PREG

11.762.5% NO ET

47.611.9%genetically

suitableEMBRYOS

35.335.3AGE

12856CYCLES

MONOGENICGENDER SELECT

MONOGENIC PGD AT MELBOURNE IVFTests developed to date:

– Huntington disease (direct)– Huntington disease (exclusion)– Cystic fibrosis – β-thalassaemia– α-thalassaemia– Duchenne muscular dystrophy– α-1-antitrypsin deficiency– Kennedy disease – Fragile–X – Motor neurone disease (exclusion)– Neurofibromatosis type 1– Hirschprung’s disease– X-linked hydrocephalus– Myotonic dystrophy– Chronic granulomatous disease– Niemann-Pick type C– Opitz syndrome– Leigh syndrome– Multiple exostosis– Rapp-Hodgkin ectodermal dysplasia– Tuberous sclerosis– X-chromosome deletion– Menkes disease– Treacher-Collins syndrome– Retinoblastoma– WHIM syndrome– Mucopolysaccharoidosis IIIB

– Congenital disorder of glycosylation– Incontinentia pigmenti– Epidermolytic hyperkeratosis– Congenital adrenal hyperplasia– Hereditary deafness DFNA17– BRCA1– Von Hippel-Lindau syndrome– Walker-Warburg syndrome– BRCA2– Familial adenomatous polyposis (FAP)– X-linked lissencephaly– Hunter syndrome

Multiple cases for many of theseMultiple cases for many of these

Tests in development:– Complex 1 deficiency– Spinal muscular atrophy (SMA)– Ataxia telangiectasia– Congenital amegakaryocytic thrombocytopenia– TTR amyloidosis– Generalised arterial calcification of infancy

(GACI)– HNPCC (hereditary non-polyposis colon

cancer

• Cystic fibrosis• Tay Sachs disease• β-thalassaemia• Sickle cell anaemia• Rh blood typing• Spinal muscular atrophy• Adrenogenital syndrome• Congenital adrenal hyperplasia• Plakophilin-1 (PKP1)• MCAD• CDG1C • Epidermolysis bullosa• Gaucher’s disease • Hyperinsulinemic hypoglycemia PHH1• Fanconis anemia• HLA matching• Fragile X• Myotonic dystrophy• Huntingtons• Wiscott-Aldrich syndrome• Incontinentia pigmenti• Ornithine transcarbamylase def.• Myotubular myopathy• Hunter syndrome• Fabry disease• Choroideraemia• Kallman syndrome• Coffin-Lowy syndrome• Barth syndrome• Hypospadias• Golabi-Rosen syndrome

• Marfans syndrome• Charcot-Marie-Tooth disease (type 1A)• Amyloid polyneuropathy• Crouzons syndrome• NF2• Osteogenesis imperfecta I and IV• Stickler syndrome• Tuberous sclerosis • Central core disease • Familial adenomatous polyposis coli• Li Fraumeni syndrome• Lesch Nyhan syndrome• Duchenne muscular dystrophy• Becker muscular dystrophy• Haemophilia A• Charcot-Marie-Tooth disease • Retinitis pigmentosa• Ornithine Transcarbamylase Deficiency• Agammaglobulinemia• Alport syndrome • Hunter’s syndrome MPSII • Oro-facial-digital syndrome type 1• Adrenoleukodystrophy• Chronic granulomatous disease• Menkes disease• Lowe syndrome• Ectodermal dysplasia• Epilepsy• BRCA1• Ataxia• Renal agenesis• Norrie disease

Conditions that have been diagnosed by PGD – worldwide

IVF Cycle Pituitary down – regulation with OCP & GNRH agonist

(gonadotrophin –releasing hormone) Ovarian stimulation with r FSH (follicle stimulating

hormone) hCG trigger Vaginal ultrasound – assisted OPU (ovum pick up) Embryo transfer (ET) 2 or 3 days after OPU

Monitor follicular maturation with vaginal ultrasound Aim for cohort of “leading follicles” of 18-20mm diameter Average egg no / OPU = 11 Fertilisation ~60%

. . . . . . . . . . . . . . . . . . . . .

Period Synarel

O C P F S H O P U E T

Acid drillingAcid drilling

Pipette loaded with acidified culture media – pH 2.4

Hole digested in zona by acid

Request for PGD submitted

PGD Committee•Senior PGD scientists•IVF doctor•Clinical geneticist•Genetics counsellor•PGD nurse

PGD refused

PGD counselling with genetics counsellor

PGD counselling with clinical geneticist

IVF counselling (mandatory in Victoria)

PGD cycle starts

ACCESS TO PGD AT MELBOURNE IVFACCESS TO PGD AT MELBOURNE IVF

CONSENT TO PGD

Although the degree of accuracy of these tests is high, all tests have a failure rate, and the test results could be wrong.

A full genetic analysis is not being carried out and there are many other genetic conditions that are not being analysed or tested for.

Finding a normal cell using FISH testing does not mean that a baby resulting from the embryo will have the normal number of chromosomes or be of the expected sex.

In single gene defect testing, we cannot guarantee that the embryo will not have the disorder being tested for.

It is strongly recommended that all women with It is strongly recommended that all women with PGD pregnancy consider DNA testing in early PGD pregnancy consider DNA testing in early pregnancy ( CVS or amniocentesis ) to confirm the pregnancy ( CVS or amniocentesis ) to confirm the early embryo diagnosis.early embryo diagnosis.

Spontaneous conception may occur during a PDG Spontaneous conception may occur during a PDG cycle, and all couples having PGD should avoid any cycle, and all couples having PGD should avoid any form of unprotected sex during the treatment cycle.form of unprotected sex during the treatment cycle.

Rarely, some embryos may be destroyed during the Rarely, some embryos may be destroyed during the biopsy procedure.biopsy procedure.

Rarely, it may not be possible to obtain a result on Rarely, it may not be possible to obtain a result on an embryo.an embryo.

Embryos that are very poor quality will not be Embryos that are very poor quality will not be subjected to embryo biopsy and will be discarded.subjected to embryo biopsy and will be discarded.

PGD - BENEFITSRELIABLE97% embryos diagnosed

ACCURATEmisdiagnosis rate ~ 2%

RAPIDembryo biopsy and diagnostic testing completed 8 – 30 hours

TREATMENT OPTIONalternative to antenatal testing and TOP

PGD – PITFALLS

InvasiveHighly medicalised, requires IVFExpensiveSpecific feasibility testing can take monthsNo guarantee of pregnancy

HIV AND ASSISTED REPRODUCTIVE TECHNOLOGY

• Chronic Viral Illness Clinic at Royal Womens Hospital Melbourne established 2002

• principle of harm minimisation (reduced risk of HIV transmission to partner and baby)

• Use of assisted reproductive technology (intra-uterine insemination or IVF)

HIV +ve MALE

.good health

.undetectable viral load (blood) for 2 months

.semen screening for HIV2 successive samples <50 copies=semen storage for IUI /IVF

(all semen samples tested for HIV RNA and DNA)

.risk of transmission to partner : <1/4000

CVI PROGRAMME RWH 2002 - 2009

~70 couples referred~30 couples treated(majority HIV-positive males)

Total number of babies born 16 inc 2 sets of twins1 miscarriage1 ectopic4 ongoing

no of patients on treatment 8no of patients pre-treatment 6

There have been no cases of transmission of HIV to partner or baby

ACKNOWLEDGEMENTSMIVF PGD TEAM

Leeanda WiltonSharyn Stock-MyerPam MatthewsMirjana MartiKay OkeAngie GiasliRebecca CameronSophie Falle

Peter ColemanDavid AmorFleur CattrallAndrea TwomeyPaisu TangCeline LawlerRiddhi MarfatiaPenelope Foster

ACKNOWLEDGEMENTSRWH CVI COMMITTEE

Michelle GilesGordon BakerHarold BourneSuellen PeakGary ClarkeSuzanne CrowePenelope Foster

Rachael Knight Jenny HoySam PernaSepehr TabriziVicky GreengrassGayle de BruyckereSuzanne Garland